Revista Brasileira de Ciências Farmacêuticas Brazilian Journal of Pharmaceutical Sciences vol. 42, n. 4, out./dez., 2006
Hernán Ricardo Lozano and Fleming Martínez*
Departamento de Farmacia, Universidad Nacional de Colombia
The ketoprofen (KTP) partitioning thermodynamics was studied Uniterms in different solvent/buffer systems such as cyclohexane (CH/W), • Ketoprofen • Partition coefficient octanol (ROH/W), isopropyl myristate (IPM/W), chloroform (CLF/ • Organic solvents W); as well as in dimyristoyl phosphatidylcholine (DMPC) and • Liposomes dipalmitoyl phosphatidylcholine (DPPC) liposome systems. In all • Transfer thermodynamics X cases, the rational partition coefficients (KO/w ) were greater than unit; therefore standard transfer free energies were negative in X sign indicating a high affinity of KTP for organic media. KO/w values were approximately eightyfive-fold higher in the ROH/W system regarding the CH/W system, thus indicating a high degree of hydrogen bonding contribution to partitioning. While in the case X of IPM/W and CLF/W systems, KO/w values were approximately only three or four-fold lower than those observed in ROH/W. On X the other hand, KO/w values were approximately seventy-five or one hundred fifty-fold higher in the liposomes compared to ROH/ W system indicating a high degree of bilayers immobilization and/ or an electrostatic contribution to partitioning. In all cases, standard transfer enthalpies and entropies of KTP from water to organic media were positive in sign indicating some degree of *Correspondence: F. Martínez participation of the hydrophobic hydration on the partitioning Universidad Nacional de Colombia processes. Finally, by using the reported data for solvation of KTP Departamento de Farmacia A.A. 14490 in water, the associated thermodynamic functions for KTP solvation Bogotá, Colombia in all tested organic phases were also calculated. E-mail: [email protected]
INTRODUCTION this drug is very scarce. As useful information in medicinal chemistry, the thermodynamics of transfer of drug Ketoprofen (KTP) is a non-steroidal anti- compounds can be studied by measuring the rational inflammatory drug widely used in the treatment of several partition coefficient as a function of temperature. Such data inflammatory and painful events (Hanson, 2000). Although can be used for the prediction of absorption, membrane KTP is widely used nowadays in therapeutics, the permeability, and in vivo drug distribution (Betageri et al., physicochemical information about transfer processes of 1996). 602 H. R. Lozano, F. Martínez
Semi-polar solvents have been found to yield better obtained values of the corresponding thermodynamic correlations with partitioning of solutes obtained in model transfer functions, an interpretation based on solute-solvent membranes compared to non-polar solvents such as interactions was developed. Therefore, the present study is cyclohexane (CH), which interacts only by non-specific a continuation of a previously presented (Lozano, forces (London interactions). In particular, octanol (ROH) Martínez, 2006). has been found to be a useful solvent as the reference for extrathermodynamic studies in a variety of systems (Leo et THEORETICAL al., 1971; Diamond, Katz, 1974; Sangster, 1997), m Isopropyl myristate (IPM) has also been used. This solvent The partition coefficient (K o/w) expressed in molality, acts as a hydrogen acceptor as well, and it has been used for any solute between organic and aqueous phases is especially for determining hydrophobic constants since it calculated by means of: simulates most closely the corneum stratum/water partition (Barker, Hadgraft, 1981; Dal Pozzo et al., 1991). IPM is (1) best related to skin/transdermal absorption because of its polar and non-polar balance simulates the complex nature where, Ww and Wo are the masses (usually in g) of aqueous
(polar/non-polar matrix) of the skin (Jaiswal et al., 1999). and organic phases, respectively, and C1 and C2 are the On the other hand, chloroform (CHL) has also been used in aqueous concentrations of solute (usually in mg mL–1) before this kind of studies since it acts mainly as a hydrogen donor and after the transfer of the solute from the aqueous phase to for establishing hydrogen bonds with solutes (Baena et al., the organic medium, respectively (Avila, Martínez, 2003). X 2004a). The rational partition coefficients (Ko/w, in mole fraction) are m However, the octanol/water system has proven to be calculated from K o/w values as: a poor model system for several drug transport processes as well as for correlating pharmacokinetic parameters. On (2) the other hand, when analyzing the behavior of several drugs, the liposome systems have shown to discriminate where, Mo and Mw are the molar masses of the organic and branched solutes regarding bulk systems such as oil/water. aqueous phases, respectively (Diamond, Katz, 1974). This has been found especially in the case of some phenols The standard change for Gibbs free energy of transfer (Rogers, Davis, 1980; Anderson et al., 1983), of a solute from an aqueous phase to an organic medium is phenotiazines (Ahmed et al., 1981, 1985), beta-blockers calculated as follows: (Betageri, Rogers, 1987), and non-steroidal anti- inflammatory drugs (Betageri et al., 1996). In addition, (3) dipyridamole partitioning was higher in liposomes than in octanol (Betageri, Dipali, 1993), as well as for mefloquine, Otherwise, the enthalpy change for the transfer may quinine, and other antimalarial drugs (Go et al., 1995, be obtained directly by thermometric titration 1997), also for some sulfonamides (Martínez, Gómez, microcalorimetry or in the case of liquid/liquid systems, 2002), and benzocaine (Avila, Martínez, 2003). For these indirectly as the difference of the respective heats of reasons, different liposome types have been used combined solution in each one of the phases, which may be obtained to organic solvents for partitioning experiments in order to by solution calorimetry (Baena et al., 2004b). As it was develop quantitative structure-activity relationships mentioned previously, a method widely used in the (QSAR studies). physicochemical study of pharmaceutical compounds As a contribution to generation and systematization (such as drugs) is by means of the analysis of the of physicochemical information about anti-inflammatory temperature-dependence for partitioning by using the van’t drugs transfer properties, the main goal of this study was Hoff method. This procedure permits obtain the standard Δ 0 X to compare the partitioning and solvation behavior of KTP enthalpy change ( Hw →ο) from: in different organic medium/buffer systems, namely: cyclohexane (CH/W), octanol (ROH/W), isopropyl myristate (IPM/W), chloroform (CLF/W), and dimyristoyl (4) phosphatidylcholine (DMPC/W) as well as dipalmitoyl Δ 0 X phosphatidylcholine (DPPC/W) liposome systems, by Therefore, Hw →ο is determined from the slope of a X employing a thermodynamic approach based on the rational pondered linear plot of ln K o/w as a function of 1/T. The partitioning variation respect to temperature. From the standard entropy change of transfer is obtained by means of: Thermodynamics of partitioning and solvation of ketoprofen 603
drug concentration by means of UV absorbance (5) measurement and interpolation on a previously constructed calibration curve for KTP in a buffer pH 7.4 Δ 0 X Δ 0 X The thermodynamic functions Hw →ο and Sw →ο (Unicam UV2-100 spectrophotometer, USA). The m/ app represent the standard changes in enthalpy and entropy, apparent molal partition coefficients (KO/ w ) were respectively, when one mole of drug is transferred from the calculated by mass balance according to Eq. (1) and m aqueous medium to the organic system at infinite dilution converted to real molal partition coefficients (KO/ w ) by expressed in the mole fraction scale (Betageri et al., 1996; using the following equation: Martínez, Gómez, 2002). (6) MATERIAL AND METHODS in which, the parenthesis term on the right side is equal to
Chemicals 1231.27 since the pH is 7.4 and the pKa of KTP corrected to an ionic strength of 0.15 mol L–1 is 4.31 (Table I). From m X In this investigation the following chemicals were KO/ w values, the rational partition coefficients (K O/ w ) were used: USP ketoprofen (US Pharmacopeia, 2004); extra calculated from the Eq. (2) employing the following molar pure grade octanol (Merck, Germany); isopropyl myristate masses: 99.48 g mol–1 for water-saturated ROH, for synthesis (Merck, Germany); cyclohexane A.R. 263.72 g mol–1 for water-saturated IPM, 113.90 g mol–1 for (Merck, Germany); chloroform A.R. (Mallinckrodt, USA); water saturated CLF, 84.16 g mol–1 for water-saturated DMPC (ref P-7331), and DPPC (ref P-5911) (Sigma CH, 18.16 g mol–1 for (ROH, IPM or CH) organic solvents Chemical Co., USA); potassium chloride A.R. (Merck, saturated buffer, and 18.62 g mol–1 for CHL-saturated Germany); sodium mono and dihydrogen phosphates A.R. buffer (Dallos, Liszi, 1995; Baena et al., 2005; Mora et al., (Merck, Germany); distilled water (conductivity < 2 µS). 2005).
Organic Solvent/Buffer Partitioning Liposome/Buffer Partitioning
Both, the aqueous and organic solvents were Liposomes were prepared by a modified Bangham mutually saturated before performing the experiments. method (1993) as it was made studying other compounds Solutions of KTP at known concentrations were (Betageri et al., 1996; Martínez, Gómez, 2002). Thin prepared in aqueous buffers adjusted to pH 7.4 at ionic films of 40 mg of DMPC or DPPC were formed on the strength of 0.15 mol L–1 (physiological values (Cevc, walls of 50 mL round-bottomed flasks after rotary 1993)). Then, in glass flasks specific volumes of organic evaporation (Buchler Instr.) of 5 mL aliquots of solvents were added to specific volumes of the aqueous chloroform solutions (8 mg mL–1). Then all flasks were KTP solutions. The employed volumes were as follows: placed in an oven at 40 °C for 2 h. Thereafter the films for ROH/W, 10 mL of ROH and 20 mL of (150 μg mL–1) were dispersed in 4 mL of KTP aqueous solution KTP aqueous solution; for CH/W, 20 mL of CH and 10 (250 μg mL–1, pH 7.4 and μ 0.15 mol L–1), then were mL of (125 μg mL–1) KTP aqueous solution; for IPM/W, heated at 30 °C (for DMPC) or 45 °C (for DPPC), and 20 mL of IPM and 10 mL of (125 μg mL–1) KTP drug vortex-mixed (Mistral® Mixer, Model 1192, Lab-Line aqueous solution; for CHL/W, 15 mL of CHL and 15 mL Instr.) until the whole film was removed from the flask of (150 μg mL–1) KTP aqueous solution. All aliquots walls. This resulted in formation of multi-lamellar were weighted in a digital analytical balance (Mettler AE vesicles (MLVs). The drug distribution was determined in 160, Germany) whose sensitivity was ± 0.1 mg. 48 hours temperature-equilibrated MLVs in 1.3 mL Mixtures were then stirred on a mechanical shaker samples, followed by centrifugation (25000 g for 60 min) (Wrist Action Burrel model 75, USA) for one hour. at the specified temperature, from UV analysis and mass Samples were placed on water baths (Magni Whirl Blue balance calculations ranging from 20.0 °C to 45.0 °C. M. Electric Company, USA) at 20.0, 25.0, 30.0, 35.0, The apparent and true partition coefficients were 40.0, and 45.0 °C (± 0.1 °C) at least for 48 hours with calculated by using the same equations as in organic sporadic stirring in order to achieve the partitioning solvent/buffer partitioning. The molar masses for lecithins equilibrium, as it was made studying other compounds are 677.90 g mol–1 for DMPC and 734.05 g mol–1 for (Martínez, Gómez, 2002). After this time-period, the DPPC. All partitioning experiments were repeated at least aqueous phase was removed followed by determining the three times and the produced data were averaged. 604 H. R. Lozano, F. Martínez
RESULTS AND DISCUSSION association or dissociation of the drug, in organic or aqueous phases (Sangster, 1997). KTP physical and chemical properties Partition coefficients of KTP The molecular structure and some physicochemical properties of KTP are summarized in table I. The pKa value Temperature-dependence of the rational partition was corrected to ionic strength (μ) of 0.15 mol L–1 (similar coefficients for the KTP in all tested partitioning systems X to that of the gastrointestinal tract and blood (Cevc, 1993)) is summarized in Table II. In all cases K o/w values are by the extended Debye-Hückel equation (Martin et al., greater than unit and increased with rising temperature. 1993). The partitioning was determined at pH 7.4 Partitioning diminishes at 25.0 °C in the following order: (resembling the blood physiological value). Such pH value DMPC/W > DPPC/W > ROH/W > CLF/W > IPM/W > was regulated with phosphate buffer having β capacity of CH/W, which is similar to data found for other drugs 0.01 calculated by the Koppel-Spiro–Van Slyke equation (Martínez, Gómez, 2002; Ávila, Martínez, 2003). Found μ (Martin et al., 1993) using pKa values corrected to data are in agreement that this drug displays a semi-polar 0.15 mol L–1. At this pH the KTP has a lower apparent and lipophilic behavior. partition coefficient value since the dissociated compound Betageri et al. (1996) has previously reported data form predominates thus having more affinity for water. regarding this drug partitioning in the ROH/W and DMPC/ This fact conduces to use Eq. (6) to obtain the true values W systems at pH 7.0 expressed as a molal scale. in order to follow the Nernst law, which implies no Nevertheless, the results presented in table II are not in
TABLE I - Physical and chemical properties of KTP
–1 λ Molecular structure M/g mol pKa máx / nm
254.28 4.45 (a), 4.31 (b) 260 (c)
(a) Reported by Fini et al. (1987) at µ 0 mol L–1; (b) Corrected at µ 0.15 mol L–1 by means of Debye-Hückel equation (Martin et al., 1993); (c) In water at pH 7.4 and µ 0.15 mol L–1
TABLE II - Rational partition coefficient of KTP in different partitioning systems as a function of temperature (± 0.05 °C). (Values in parenthesis are standard deviations)
X System K o/w 20.00 °C 25.00 °C 30.00 °C 35.00 °C 40.00 °C 45.00 °C CH/W 61 82 104 142 175 297 (5) (5) (8) (8) (8) (23) ROH/W 6850 7069 7321 7690 8250 9510 (50) (20) (27) (70) (50) (70) IPM/W 1670 1760 1860 2130 2350 2700 (50) (70) (50) (70) (70) (110) CLF/W 2100 2210 2320 2500 2920 3170 (60) (80) (60) (110) (50) (60) DMPC/W 289000 1041000 1402000 1841000 2469000 3400000 (17000) (42000) (40000) (22000) (54000) (110000) DPPC/W 382000 541000 751000 1088000 1695000 5872000 (22000) (19000) (24000) (22000) (13000) (95000) Thermodynamics of partitioning and solvation of ketoprofen 605 agreement with those presented in literature when they were liposomes are in a fluid state (liquid crystal) while DPPC- converted to mole fraction. Particularly, for the DMPC/W liposomes are in a rigid state (gel) since the transition gel- system according to Betageri et al. (1996), it was found liquid crystal temperature (TC) for these compounds are X diminished K o/w values when increasing the working 23.6 (± 1.5) °C and 41.3 (± 1.8) °C, respectively (Koynova, temperature, which is on the contrary with data presented Caffrey, 1998). Therefore, liposomes flexibility and in the present study. On the other hand, accordingly to permeability is greater in a fluid state than in a rigid state, graphical results presented in the same reference (Betageri i.e. at temperatures higher than TC. et al., 1996), apparently the KTP molal partition is similar On the other hand, when partitioning is compared in both ROH/W and DMPC/W systems, which is not in for liposomes present in the same state, partitioning is agreement to Table II data even if partition coefficients are higher for DPPC/W respect to DMPC/W, i.e. at 20.0 °C converted to molality (Lozano, Martínez, 2006). both liposome systems are in a gel state (rigid) while at According to Table II, the partitioning in DMPC is 45.0 °C both liposome systems are in liquid crystal state almost one hundred fifty-fold greater than its obtained (flexible). In particular, this result could be considered value in ROH/W at 25.0 °C. When comparing the results as an additional demonstration about the lipophilic obtained for all organic solvents, it can be deduced a nature of this drug since DPPC has two additional higher preference of KTP for octanol, respect to the other methylene groups in each hydrocarbon moiety with tested solvents, in particular regarding cyclohexane. As respect to DMPC. Nevertheless, certainly KTP is not a has been previously described in literature (Sangster, hydrophobic drug because CH/W partitioning is 1997; Mora et al., 2005), ROH has a microheterogeneus relatively low when compared to that obtained in the structure on water saturation. For this reason, ROH other organic solvents. In this point, it is very important interacts with KTP by hydrogen bonding thorough to remark that it will be very important to analyze the carbonyl and carboxyl groups present in this drug, in surface behavior of this drug in other organic systems addition to weak interactions, such as London dispersion such as micelles, in order to evaluate if KTP is forces, which conduce to structural immobilization of amphiphilic in nature, as it was demonstrated for drug molecules near to the alkyl moieties of octanol. ibuprofen by Stephenson et al. (2006). As it was already discussed, the phospholipidic vesicles have been investigated as model for studying Seiler and other analogue parameters drug distribution in membranes, since liposomes resemble the ordered structures present in biological Seiler (1974) proposed an equation analogue to Eq. membranes. As it is shown in Table II, partitioning is (7) in order to compare partition coefficients of a drug in the higher in these systems compared to ROH/W. This fact ROH/W and CH/W systems, with the basic purpose of demonstrates a high contribution to partitioning by the obtaining information related to contribution of hydrogen drug immobilization inside the bilayers, in addition to bonding for partitioning of solutes. In a more complete electrostatic interactions with the polar head of treatment, other considerations such as molecular geometry phospholipids as has been described in the literature and steric effects of solutes and solvents should be (Ahmed et al., 1981, 1985; Betageri, Rogers, 1987; considered for such aim. However in a first approach, Eq. Betageri, Dipali, 1993; Abe et al., 1995). In addition, the (7) is a good attempt for identifying the main solute-solvent vesicles can immobilize the drug in the space available interactions affecting the solute transfer: within the bilayers increasing the KTP solubilization. On the other hand, some surface phenomena due to the (7) possible amphiphilic nature of KTP would be also considered because the interfaces’ nature would be The above equation may also be written as a quotient modified. This consideration has been done recently by Δ X X X relationship in the form log K ROH/W = log (K ROH/W /K CH) studying the behavior of ibuprofen in micellar solutions showing the hydrogen bonding nature of the interactions of ionic and non-ionic surfactants (Stephenson et al., between the drug and octanol with respect to cyclohexane. 2006). Δ X If log K ROH/W is greater than 0, then this result indicates Comparing the results obtained in the liposome some contribution of hydrogen bonding to the partitioning system, the partitioning is higher in DMPC/W with respect of drugs. Table III presents the values of parameter of Seiler to DPPC/W ranging from 25.0 to 40.0 °C. Such a behavior and other analogous parameters for KTP at 25.0 °C, could be explained in terms of the respective aggregation calculated from the different rational partition coefficients states of liposomes. That is, in this range the DMPC showed in table II. 606 H. R. Lozano, F. Martínez
TABLE III - Seiler and other analogue parameters of KTP at 25.00 ± 0.05 °C
X X Δ X Parameter System 1 System 2 log K o/w (Syst1) log K o/w (Syst2) log K o/w (a) Δ X log K ROH/W ROH/W CH/W 3.849 1.915 1.935 Δ X log K IPM/W IPM/W CH/W 3.245 1.915 1.331 Δ X log K CLF/W CLF/W CH/W 3.344 1.915 1.429 Δ Xα log K ROH/W ROH/W IPM/W 3.849 3.245 0.604 Δ Xβ log K R OH/W ROH/W CLF/W 3.849 3.344 0.505 Δ X log K DMPC/W DMPC/W ROH/W 6.017 3.849 2.168 Δ X log K DPPC/W DPPC/W ROH/W 5.733 3.849 1.884 ( )Δ X X X a log K o/w = log K o/w (Syst1) - log K o/w (Syst2)
CH is an aprotic solvent unable to form hydrogen (hydrogen acceptor) in KTP are the carbonyl group present bonds as donor or acceptor, and therefore acts only through between two aromatic rings and the carbonyl moiety in the non-specific interactions (London forces). However carboxyl group. At this point, it is convenient to remind that hydroxyl group of ROH can be an acceptor and/or a donor in the previously made analyses we consider only the effect of protons, and moreover, as was already expressed, its of hydrogen bonding without consider other kind of alkyl moieties allow the structural immobilization of solutes interactions or geometric parameters, such as differences in due to the tetrahedral microstructure adopted in saturation molecular sizes. by this solvent in contrast to the CH behavior (Sangster, More recently, Eq. (8) has been exposed in order to Δ X 1997; Mora et al., 2005). log K ROH/W includes obtain information about the higher distribution of drugs contributions by hydrogen bonding and by structural inside liposome bilayers compared to that obtained in immobilization to the partitioning (in this analysis is octanol (Avila, Martínez, 2003). considered that the non-specific interactions are similar for all organic solvents and drugs). (8) Δ X log K IPM/W allows to estimate the contribution of the Δ X solvent as hydrogen bonding acceptor in IPM/W rational The log K DMPC/W parameter allows estimating the partitioning. By comparison of Seiler parameter contribution to partitioning by structural immobilization Δ X Δ X ( log K ROH/W) with log K IPM/W it shows that the octanol, and by solute-lipid electrostatic effects depending on the besides of contributing to the drug partitioning as acceptor phospholipid employed for preparing of liposomes; that is, of hydrogen, may also contribute as hydrogen donor, depending on the pH, the alkyl moiety length, and the Δ X therefore log K IPM/W value is smaller than the Seiler possible presence of ionic charges in the phospholipids’ Δ Xα parameter. A third parameter, namely: log K ROH/W was polar heads, thus the results may be variable (Ahmed et al., calculated comparing the ROH/W and IPM/W partition 1981, 1985; Betageri, Rogers, 1987; Betageri, Dipali, coefficients in order to establish the contribution of the 1993). Table IV shows that the effect due to immobilization organic solvent as hydrogen donor to the partitioning. As it is higher in DMPC liposomes than in DPPC liposomes. was already said, CHL acts mainly as hydrogen donor and This behavior can be explained in terms of the liposome therefore, other two parameters were calculated in order to aggregation state according to the temperature. As was analyze the contribution of this kind of interaction on the already exposed, in the studied range the DMPC liposomes Δ X partitioning of KTP. log K CLF/W permits to observe the are in a liquid crystal state (fluid), whereas the DPPC possible contribution of CLF as hydrogen donor, while liposomes are in a gel state (rigid). Δ Xβ log K R OH/W (obtained from ROH/W and CLF/W) on the other side permits to evaluate the behavior of ROH as KTP Partitioning Thermodynamics hydrogen acceptor. Δ X Generally, the results show greater log K o/w values Van’t Hoff plots for CH/W, ROH/W, IMP/W and Δ X when the drug acts as an hydrogen acceptor ( log K CLF/W CLF/W partitioning of KTP are shown in figures 1, 2 and Δ Xα and log K ROH/W) respect to those obtained when the drug 3, respectively. For CH/W system (Figure 1) a lineal Δ X Δ Xβ acts as hydrogen donor ( log K IPM/W and log K ROH/W ). The regression model was developed without consider the acid hydrogen present in this drug is the one present in its obtained value at 45.0 °C, because deviates from the carboxylic group. On the other hand, the basic groups tendency followed by the other five points. The obtained Thermodynamics of partitioning and solvation of ketoprofen 607 determination coefficient was 0.979. For the ROH/W Figure 4 shows the van’t Hoff plot for KTP system (Figure 2) a parabolic regression model was partitioning in DMPC and DPPC liposomes. In both ca- developed without consider the value at 45.0 °C, because ses the results were adjusted to lineal regression models it also deviates from the tendency followed by the other considering the temperature intervals for which the stated points. In this case, the determination coefficient was liposomes are in the same aggregation state. 0.993. For IPM/W and CLF/W systems (Figure 3), Determination coefficients were 0.995 and 0.991, for parabolic regression models were developed considering all DMPC and DPPC respectively. As it was already the obtained data. Determination coefficients were 0.968 explained, transition temperatures for DMPC and DPPC and 0.961, respectively. liposomes are 23.6 and 41.3 °C respectively (Koynova, Caffrey, 1998). Therefore, temperature intervals considered were from 25.0 to 45.0 °C for DMPC (fluid state) and from 20.0 to 40.0 °C for DPPC (rigid state).
FIGURE 1 - Van’t Hoff plot for the rational partitioning of KTP in the CH/W system.
FIGURE 4 - Van’t Hoff plot for the rational partitioning of KTP in the DMPC (circles) and DPPC (squares) liposome systems. Vertical discontinued lines are the phase transition temperatures for liposomes: 23.6 °C and 41.3 °C, for DMPC and DPPC, respectively.
From the estimated slopes in the CH/W, DMPC/W and DPPC/W systems, the respective standard enthalpic changes for transfer were calculated by using Eq. (4) using methods of error propagation (Bevington, 1969). In the case of ROH/W, IPM/W and CLF/W systems, in which FIGURE 2 - Van’t Hoff plot for the rational partitioning of parabolic regressions were obtained, the first derivatives KTP in the ROH/W system. were taken for these models and resolved at 25.0 °C. Then, Δ 0 X transfer enthalpies ( Hw →ο) were calculated as the product of slopes multiplied by –R (that is, –8.314 J mol–1 K–1). The obtained values for this thermodynamic function are summarized in table IV, in addition to standard Gibbs free Δ 0 X energy for transfer ( Gw →ο) of KTP from water to different organic systems (expressed in mole fraction at 25.0 °C). Δ 0 X The Gw →ο values were calculated by means of Eq. (3) based on partitioning data of Table II using methods of Δ 0 X errors propagation (Bevington, 1969). In all cases, Gw →ο was negative in sign indicating the preference of this drug for organic media. Δ 0 X Δ 0 X On the other hand, from Gw →ο and Hw →ο values, FIGURE 3 - Van’t Hoff plot for the rational partitioning of the respective standard entropic changes of transfer Δ 0 X KTP in the CLF/W (circles) and IPM/W (squares) systems. ( Sw →ο) in mole fraction were calculated from Eq. (5). 608 H. R. Lozano, F. Martínez
Δ 0 X Δ 0 X Δ 0 X TABLE IV - Standard Gibbs free energy ( Gw →ο), enthalpy ( Hw →ο), and entropy ( Sw →ο) of KTP transfer from water ζ ζ to different organic systems. Relative contributions of enthalpy ( H) and entropy ( TS) toward transfer processes at 25.00 ± 0.05 °C. (Values in parenthesis are standard deviations)
Δ 0 X Δ 0 X Δ 0 X Δ 0 X ζ ζ System Gw →ο / Hw →ο / Sw →ο / T Sw →ο / H TS kJ mol–1 kJ mol–1 J mol–1 K–1 kJ mol–1 CH/W –10.93 40.9 174 51.8 44.1 55.9 (0.14) (1.7) (7) ROH/W –21.97 4.94 90 26.9 15.5 84.5 (0.01) (0.25) (5) IPM/W –18.52 9.3 34.8 10.4 47.2 52.8 (0.10) (0.6) (2.4) CLF/W –19.09 7.8 29.2 8.7 47.2 52.8 (0.09) (0.6) (2.1) DMPC/W –34.35 46.3 270 80.6 36.5 63.5 (0.10) (0.9) (5) DPPC/W –32.72 56.2 298 88.9 38.7 61.3 (0.09) (1.5) (8)
Δ 0 X These values are also presented in Table IV. The Hw →ο On the other hand, after than a certain number of values presented in Table IV are almost the same obtained solute molecules have migrated from the aqueous to the for CH/W, ROH/W, DMPC/W and DPPC/W partitioning organic phase, the original cavities occupied by the drug in expressed in molality (Lozano, Martínez, 2006) because the aqueous phase have been now occupied by water this property is not dependent on the concentration scale molecules. This event produces an energy releasing due to Δ 0 X used (Katz, Diamond, 1974). On the other hand, Gw →ο water-water interactions. However, depending on solute’s Δ 0 X and S w →ο values are different with respect to those molecular structure, it is also necessary to keep in mind the obtained using molality because they imply the possible disruption of water structure, that is, the water concentration scale used in calculating partition molecules organized around the alkyl or aromatic groups of coefficients. Moreover, the considered reference states are drug (hydrophobic hydration). This event in particular different in both cases, that is, ideal solutions of drug implies an intake of energy in addition to a local entropy having unit mole fraction in each phase (solute pure) in the increase by separation of some water molecules which former case, and ideal solutions of drug having 1.0 mol originally were associated among them by hydrogen Kg–1 in each phase in the later case. bonding (Tanford, 1973). The enthalpic and entropic changes imply respec- From Table IV it can be observed that for all cases, tively, the energetic requirements and the molecular the transfer processes from water to organic media were randomness (increase or decrease in the molecular endothermic, which imply high increments in the systems’ disorder), implied in the net transfer of the drug from water net entropy. In principle, it could be said for the CH/W to different organic media. In general terms, it should be system, that the high values in enthalpy and entropy are due considered independently the behavior presented in each mainly to disruption of water-icebergs present around the phase, before and after the partitioning process. hydrocarbon groups of this drug (two aromatic rings and Since initially the solute is present only in water, one methyl group), and on the other hand, the creation of a then, it is necessary to create a cavity in the organic cavity in the cyclohexane to accommodate the solute. Both medium in order to accommodate the solute after events, as was already said, imply an energy intake and a transfer. This is an endothermic process, since an energy disorder increase at molecular level. supply is necessary to separate the organic solvent In the case of ROH/W system, the enthalpy of molecules. When the solute molecules are acco- transfer is relatively low, while the entropy of transfer has mmodated in the organic phase an amount of energy is a middle value. These values could be explained in terms of released due to solute-organic solvent interactions. This a possible organization in the water-saturated octanol due event implies an entropy increase in this medium due to to the replacement of an octanol molecule by one of drug. mixing process. This replacement would be present in some of centers Thermodynamics of partitioning and solvation of ketoprofen 609
conformed by two water molecules and four octanol Based on data for%zH and%zTS from table IV it molecules, inside the microheterogeneus structure of this follows that transfer of KTP from water to all organic water-saturated organic solvent (Sangster, 1997; Mora et systems is driven mainly by organizational changes. This al., 2005). The previous event releases energy and confirms fully that previously observed for the signs of compensates the molecular disorder produced by the drug- entropies of transfer, which are positive in all cases, that is, organic solvent mixing process. the partitioning is entropy driven because of enthalpies are For the IPM/W and CLF/W systems, the enthalpies also positive in all cases. Although in the case of IPM/W and entropies of transfer are relatively low compared with and CLF/W almost equivalent contributions of enthalpy the other systems. Because, some information about the and entropy toward the transfer process is supposed. The structural properties for these water-saturated organic ROH/W system has the higher entropy contribution toward solvents is not available at the moment, it is not possible to transfer followed by liposomes. As has been already said, explain these results at molecular level. the dominant effect of entropy on Gibbs free energy of On the other hand, in the case of liposomes, besides of transfer from water to ROH would be due to disorder that previously described for the aqueous media in the CH/ increase presented in the micro-heterogeneous structure of W system (which may be also valid for the ROH/W, IPM/W water-saturated octanol by the solute accommodation and CLF systems), because of the highly organized structure (Sangster, 1997; Mora et al., 2005). of phospholipidic bilayers, the energy requirement for separate the bilayers, in order to accommodate the solute Thermodynamics of KTP solvation molecules, implies a relatively high enthalpy of transfer. The same event implies a high increment in the systems’ entropy According to Katz and Diamond (1974), the values Δ 0 X by the disorder generate inside the vesicle bilayers. of thermodynamic functions of partitioning, G w →ο , Δ 0 X Δ 0 X By comparing the values obtained for both Hw →ο and Sw →ο, depend both upon interactions between thermodynamic properties, it follows that they are higher drug and water and upon interactions between drug and for DPPC liposomes (rigid state), than those for DMPC organic medium. In order to obtain quantities that can be liposomes (fluid state). This result would be explained in discussed solely in terms of drug-organic medium terms of the lower flexibility of DPPC liposomes with interactions, the contributions of drug-water must be respect to DMPC liposomes, and therefore, the higher removed. This can be accomplished by referring to energy requirements for accommodate the solute inside the hypothetic processes presented in Figure 5: DPPC bilayers. This behavior is similar to that presented by the liposome partitioning of any other drugs, such as benzocaine and some sulfonamides (Martínez, Gómez, 2002; Ávila, Martínez, 2003). Equations (9) and (10) were used in order to evaluate the respective contributions in absolute values for enthalpy and entropy toward the standard Gibbs free energy of transfer and indeed to identify the dominant effect on transfer, that is, energy changes or molecular organization changes. Similar equations have been used recently in order FIGURE 5 - Transfer processes of KTP between water, to evaluate the relative contributions by enthalpy and organic medium, and vapor phase. entropy to the free energy of solvation of some non- steroidal anti-inflammatory drugs studying the solution In this scheme (Figure 5), ΨOX, stands for any process in several solvents (Perlovich et al., 2003, 2004). thermodynamic function whose change can be measured The respective contributions for all the partitioning systems when one mole of drug is transferred between water, ΔΨ OX evaluated are also presented in table IV. organic medium and the vapor phase. The term w represents the standard Gibbs free energy, enthalpy, or ΔΨ 0X entropy of solvation of drug in water, while the term O (9) represents correspondingly the standard Gibbs free energy, enthalpy, or entropy of solvation of drug in organic medium. From this, the following equations can be stated: (10) (11) 610 H. R. Lozano, F. Martínez
(12) positive values at molecular level is unclear. According ζ ζ to H and TS values, the enthalpy is the main property (13) contributing to solvation process in all media, including the aqueous phase. ΨOX where, v is the respective thermodynamic value of the In order to clarify and understand the specific ΔΨO X function in the vapor phase. The w→o values for KTP interactions presented between KTP and DMPC and obtained from partitioning experiments are presented in DPPC liposomes it would be important to dispose ΔΨO X table IV. On the other hand, the w values of solvation information about UV, IR and NMR spectral data, and of this drug in water have been presented by Perlovich et al. DSC calorimetry, among others. These instrumental ΔΨO X (2003). From these values, the o values were techniques have been used in other studies about calculated by means of: solubility and partition of other drugs in aqueous, organic, and liposomal systems. Some properties of (14) pharmaceutical compounds studied have been the solubility of parabens in dioxane-water mixtures Table V shows the standard thermodynamic (Shihab et al., 1971), the interaction of propranolol with functions of solvation of KTP in water presented by liposomes (Surewics, Leyko, 1981), the interaction of Perlovich et al. (2003). The same table present also the phenylbutazone in DMPC and DPPC liposomes (Sainz standard thermodynamic functions of solvation of this et al., 1993), and the interaction of paclitaxel with drug in all organic phases studied, including the DPPC liposomes (Zhao et al., 2004), among several respective contributions by enthalpy and entropy toward others. solvation in each medium (calculated by means of expressions analogous to equations (9) and (10)). In all CONCLUSIONS Δ 0X Δ 0X cases, the Gsolv and Hsolv values are negative. These results indicate the preference of KTP by organic media From the previously exposed analysis, in general respect to its vapor phase, and also indicate that the terms it could be concluded that KTP has mainly a Δ 0X solvation process is enthalpy driven. In the case of Ssolv, lipophilic behavior although this drug is not certainly a this function is negative for organic solvents, which hydrophobic drug. On the other hand, this drug possess implies a diminishing in the molecular randomness by a greater affinity by liposomes with respect to all passing of drug molecules from vapor state to liquid evaluated organic solvents, which confirm the original media. On the other hand, for liposomes this function idea about the greater discriminating power of liposomal has positive values which indicate a net increase in vesicles in QSAR studies. Finally, it will be very disorder after drug-lecithins interactions which overlap important to analyze the surface behavior of this drug in the diminishing in molecular randomness by passing of other organic systems such as micelles, in order to drug molecules from vapor state to condensate state evaluate if KTP is amphiphilic in nature, as it was inside the liposomes. Nevertheless, the meaning of these demonstrated for ibuprofen.
Δ 0 Δ 0 Δ 0 TABLE V - Standard Gibbs free energy ( Gsolv), enthalpy ( Hsolv), and entropy for solvation ( Ssolv) of KTP in water and ζ ζ several organic systems. Relative contributions of enthalpy ( H ) and entropy ( TS ) toward solvation processes at 25.00 ± 0.05 °C
Δ 0 Δ 0 Δ 0 Δ 0 ζ ζ System Gsolv / Hsolv / Ssolv / T Ssolv / H TS kJ mol–1 kJ mol–1 J mol–1 K–1 kJ mol–1 W (a) –34.20 –92.88 –196.8 –58.74 61.3 38.7 CH –45.13 –52.0 –23 –6.9 88.3 11.7 ROH –56.17 –87.94 –107 –31.8 73.4 26.6 IPM –52.72 –83.6 –162.0 –48.4 63.4 36.6 CLF –53.29 –85.1 –167.6 –50.0 63.0 37.0 DMPC –68.55 –46.6 74 21.9 68.1 31.9 DPPC –66.92 –36.7 101 30.1 54.9 45.1 (a) Reported by Perlovich et al. (2003). Thermodynamics of partitioning and solvation of ketoprofen 611
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